1 Intestinal fermentation: dietary

and microbial interactions

A. Pivaa, F. Galvanob, G. Biagia and G. Casadeia

a
DIMORFIPA, Universita degli Studi di Bologna, Via Tolara di Sopra 50,
40064 Ozzano Emilia, Italy
b
Department of Agro-Forestry and Environmental Science, Mediterranean
University of Reggio Calabria, Piazza S. Francesco 7, 89061 Reggio
Calabria, Italy

The gastrointestinal tract of growing animals represents a complex and constantly changing
milieu, according to the result of complex interactions between dietary ingredients (influ-
enced by their chemical and physical characteristics), age, production stage and immune
status of the animal, environment management and microflora metabolism. The antibiotic
growth promoter era is at its endpoint and new strategies to maintain high and safe production
standards are needed. In this scenario, no longer bacterial inhibition, but rather bacterial mod-
ulation should be the primary target of all research efforts. Moreover, any alternative to
antibiotics should be properly studied and must fit to production conditions and market
requirements in order to be successful. Addition of organic acids, prebiotics and probiotics, as
well as lowering the dietary buffering capacity and direct feeding of specific nutrients to
sustain intestinal mucosa functions, are all strategies that require in-depth investigation. Some
efforts are in progress to assess the advantages of combo strategies where, for example, a
blend of organic acids could cumulate the effects of the different acids on animal physiology and
microbial metabolism, while a symbiotic combination could maximize the efficacy of a prebi-
otic NDO (nondigestible oligosaccharide) by coupling it with a probiotic strain that can
electively ferment it. Science in the post-antibiotic era of animal farming is facing an intriguing
challenge that will give a successful return only if applicable and reliable in practical situations.

1. INTRODUCTION
The growth-promoting effects of antibiotics in animal diets have been well established for over
50 years, ever since Stokstad and Jukes (1949) demonstrated that the presence of tetracycline
residues in poultry feeds increased the growth of the animals. Improved performances follow-
ing the use of therapeutic antimicrobials were then described in turkeys (Stokstad and Jukes,
1950), pigs (Jukes et al., 1950), and ruminants (Jukes and Williams, 1953; Stokstad, 1954).

Biology of Nutrition in Growing Animals

.
R. Mosenthin, J. Zentek and T. Zebrowska (Eds.)
3 2006 Elsevier Limited. All rights reserved.
4 A. Piva et al.

The major benefits derived from the use of antibiotics in subtherapeutic doses in animal
feeding involve: disease prevention, improved feed efficiency and increased performances,
especially for the young stressed animals and where management and hygiene conditions are
not excellent. In pig farming, feeding antibiotics is widely practiced around weaning, the time
that represents the most challenging period a pig encounters during its life in terms of infec-
tion and abundance of stressors. In older pigs raised for slaughter, the use of feed antibiotics
is generally regarded as unnecessary and not cost effective. Feed antibiotics have occasionally
been shown to reduce the number of bacteria present in the gut (Jensen, 1988) but more often
they appear to have little effect on total counts of viable bacteria.
Although the mechanism by which antibiotics promote growth is still under heated debate,
the most reliable hypothesis relates to changes in the composition of the intestinal microflora.
Walton (1983) identified six possible different modes of action for growth promoting agents:
(1) the production of discrete lesions in the cell wall of enteric bacteria; (2) a reduction in the
thickness of the intestinal mucosa; (3) an increase in intestinal alkaline phosphatase levels;
(4) a reduction in amounts of bacterial toxins and toxic metabolites produced in the intestine;
(5) a decrease in the level of production of intestinal ammonia; and (6) an energy-sparing effect.
The development of antimicrobial resistance over the last four decades has led to an inten-
sification of discussions about the prudent use of antimicrobial agents, especially in veterinary
medicine, animal nutrition and agriculture. One common outcome has been the conclusion
that the use of antimicrobial drugs and the development of resistance in human and animals
are interrelated and that systems should be established to monitor antimicrobial resistance in
pathogenic and commensal bacteria of animal origin.
The magnitude of antibiotic usage in agriculture is pretty impressive. As reported by Witte
(1998), in Denmark during the year 1994, a total of 24 kg of vancomycin were used for human
therapy compared to 24 000 kg of the similar antibiotic, avoparcin, in the animal industry. It has
to be noted that vancomycin and avoparcin have a common mode of action, which greatly
increases the danger of developing cross-resistance in bacteria. As reported by the DANMAP 2002
data, after antimicrobial growth promoters (AGP) were banned in 1998, Danish usage of thera-
peutic antimicrobials increased (+68%) from 57 300 kg of active compound in 1998 to 96 202 kg
in 2001, but total consumption of antimicrobials in food animals decreased by more than 50%.
In 1969, the Swann Committee of the United Kingdom concluded that antibiotics used
in human chemotherapy or those that promote cross-resistance should not be used as growth
promoters in animals, in order to reduce the risk of spreading antibiotic resistance. This
recommendation led to the subdivision of antibiotics into two main categories: those for dietary
use, requiring no prescription and those for medical use, requiring medical prescription.
In 1985, Sweden decided to allow the use of feeds containing antibiotics or other
chemotherapeutic substances only via veterinary prescription and on a case-by-case basis.
Tylosin and virginamycin (banned in the EU since January 1, 1999) have been recently
shown to induce cross-resistance to antibiotics used in human therapy (Jacobs, 1997; Witte,
1998), while other significant examples of induction of microbial resistance were reported at
the WHO meeting in Berlin in 1997 (WHO, 1997).
Although the major cause of resistance to antibiotics in human pathogens is medical
prescription usage of these drugs, the concerns about the spreading of antibiotic resistance
culminated, as of January 1, 1999, in a ban of the use of most antibiotics utilized as growth
promoters, such as bacitracin, tylosin, spiramycin, virginamycin, olaquindox and carbadox.
Avoparcin had already been banned since April 1, 1997, after it was realized that enterococci
isolated from the intestine of chickens and pigs fed avoparcin were resistant to vancomycin
(Bager et al., 1997), an antibiotic commonly used in human therapy.
 Intestinal fermentation: dietary and microbial interactions 5

The reduced use of antibiotics would be expected to cause a progressive reduction in

acquired resistance and the micro-organisms with acquired resistance should be less viable
and, with reduced antibiotic-induced pressure, should be progressively eliminated by the
ecosystem. However, Morrel (1997) showed that some antibiotic-resistant strains of
Escherichia coli have evolved compensatory mutations that preclude reversion to the sensitive
state, even without selective pressure.
Considering the intention of organizations and the EU to end all use of antibiotics as growth
promoters by 2006, the need for novel strategies to modulate the gastrointestinal environment
and microflora metabolism is of top priority.

2. STOMACH
2.1. Microflora

After birth, piglets have to rapidly adapt to significant nutritional and environmental changes
throughout the postnatal and weaning periods. More precisely, this adaptation involves the
gastrointestinal tract, with its digestive, fermentative, absorptive and immunological func-
tions, as these functions will affect the health status and production performance in the
subsequent periods (Pluske et al., 1997).
At birth, the intestinal tract of pigs is sterile (Sinkovics and Juhasz, 1974) and represents a
good niche for rapid proliferation of environmental bacteria. Lactobacilli, streptococci,
coliforms and clostridia are the main bacterial groups that can be isolated from gastric content
within the first 23 hours of life. The major source of bacteria for the newborn pig is mater-
nal feces. Furthermore, the piglet also acquires bacteria during birth from the sows fecally
contaminated vagina and perineum, as well as from the frequent contact with the sows
contaminated skin (Arbuckle, 1968).
The stomach is the first good site for bacterial proliferation due to the low flow rate of
digesta and the nutritionally rich content present in it. Lactobacilli and streptococci can fer-
ment milk lactose, and they increase numerically very rapidly during the first 2448 h, and
remain the dominant stomach population for the following suckling period. At this time
microbial cell concentrations reach values of 107109 per gram of gastric content (Jensen,
1998). As the main metabolic product of lactic acid bacteria is lactic acid, the pH drops
consistently (34) inhibiting the proliferation of other bacteria (Jensen, 1998).
A number of variables such as nutrient availability, type of feed introduced, flow of digesta,
pH and dry matter content, may have effects on gastrointestinal microbial diversity. At wean-
ing, dietary shifts from a liquid to a solid feed determine a dramatic rearrangement of
microbial populations. Jensen (1998) showed that at weaning time the previously dominating
lactobacilli leave more space to coliforms, whose plate counts seem to be higher at day 2 and
4 postweaning. This seems to be a temporary pattern that goes back to normality (higher lactic
acid bacteria and lower coliforms) one week after weaning. This kind of variation, coupled
with the different stressors (regrouping, sow withdrawal, etc.), make animals more sensitive
to possible microbial imbalance and susceptible to scours.

2.1.1. Microbial metabolism

Establishment of appropriate microflora at this time is of particular interest with respect to

gastric pH maintenance. Cranwell et al. (1976), in their observations on gastric content and
fermentation, reported that HCl secretion in suckling piglets is rather low because of mucosa
6 A. Piva et al.

immaturity and low feed stimuli. Lactic acid, produced in an almost inverse relationship
to HCl, stabilizes pH values around 34, which is high enough to permit lactic acid bacteria
proliferation and fermentation of sows milk lactose. The final pH reached under these condi-
tions, together with maternal immunity are sufficient to depress growth of other potentially
dangerous bacteria. A different pattern is likely to occur at weaning when market conditions
force pig producers to reduce the natural weaning age (1319 weeks) down to 34 weeks.
In fact, weaning pigs at 34 weeks exposes animals to nutritional, environmental and social
stressors that usually result in a postweaning phase characterized by low weight gain, low feed
intake and diarrhea (Barnett et al., 1989). Blechea et al. (1983) reported decreased cellular
immunity in pigs weaned at 23 weeks of age, whereas cellular immunity was not altered by
weaning pigs at 5 weeks. At this age the immunological status of a piglet is also low, as pas-
sive immunity acquired through maternal colostrum is dramatically decreased, and active
immunity is only just beginning to develop (Gaskins and Kelly, 1995). This postweaning lag
period may be related to insufficient secretions of gastric acid or pancreatic amylase, lipase
and trypsin (Kidder and Manners, 1978).
Acid secretion in young pigs does not reach appreciable levels until 34 weeks after wean-
ing (Cranwell and Moughan, 1989). The suckling pig uses two strategies to counteract the
limitation of insufficient acid secretion and these have been discussed by Easter (1988). The
primary strategy involves the conversion of lactose in sows milk into lactic acid by the lactic
acid bacteria residing in the stomach. Secondly, the nursing pig reduces the need for transitory
secretion of copious amounts of acid by frequent ingestion of small meals.
Failure to maintain a low gastric pH has important implications for the digestive functions
of the early-weaned pig. An elevated pH would cause a reduction in the activation of pepsino-
gens, which occurs rapidly at pH 2 and very slowly at pH 4 (Taylor, 1962). Pepsins have two
pH optima, 2 and 3.5, and their activity declines above 3.6, with no activity at pH > 6.0
(Taylor, 1959). As a result, feed proteins may enter the small intestine almost intact. Since the
end-products of pepsin digestion also stimulate the secretion of pancreatic proteolytic
enzymes (Rerat, 1981) an increased gastric pH may indirectly contribute to lower pancreatic
secretion with an eventual reduction in the efficiency of protein digestion.
Inefficient digestion may also provide a basis for the initiation of scours in the young pig
because of the provision of abundant undigested substrates in the small intestine to support
the proliferation of coliforms.
An acid gastric environment is believed to have pronounced bactericidal properties for cer-
tain micro-organisms, in particular for the Enterobacteriaceae (Sissons, 1989), whilst lactic
acid bacteria can still play their beneficial role under such conditions. Viable micro-organisms
entering the digestive tract via the mouth need to pass through the acidic conditions of the
stomach to successfully colonize the small intestine. A rise in gastric pH would, therefore,
allow increased proliferation of Enterobacteriaceae, including Escherichia coli (Smith and
Jones, 1963), which has been associated with scours and increased mortality (White et al.,
1969; Thomlinson and Lawrence, 1981). Furthermore, evidence suggests that proliferation of
coliforms in the stomach may lead to a further decrease of gastric acid secretion due to the
release of a bacterial polysaccharide with an inhibitory effect on acid secretion (Baume et al.,
1967; Wyllie et al., 1967). Uehara et al. (1990, 1992) found that bacterial lipopolysaccharide
(LPS) or endotoxin in minute doses inhibits the secretion of gastric acid and pepsin in rats.
The results showed there was a dose-dependent decrease of gastric acid secretion in rats after
intraperitoneal injections of LPS (101000 ng/rat). Subsequent histological analysis did not
reveal any mucosal or parietal cell lesions, excluding a toxic mode of action of the
lipopolysaccharide. Moreover, 24 h after injection, basal acid output returned to normal levels,
 Intestinal fermentation: dietary and microbial interactions 7

indicating a reversible action. Tsuji et al. (1992) observed that the effect of Escherichia coli
lipopolysaccharide was blocked by indometacin, suggesting that LPS needs an intact
prostaglandin system to exhibit its inhibitory action on gastric secretion.

2.2. Buffering capacity

During suckling, the buffering mechanisms affecting gastric pH, mainly saliva, bicarbonate
and mucus secretions are not a major problem for the piglets. At weaning however, when ani-
mals begin to consume solid feed and water is drunk ad libitum, the buffering capacity of the
diet represents a major obstacle.
In order to describe the ability of a diet to buffer HCl secretions and cause a high gastric
pH, several authors have measured the acid-binding capacity (ABC) of the feed. In this case
ABC is defined as the amount of acid in milliequivalents (mEq) required to lower the pH of
1 kg of feed to pH 4 (ABC-4) or pH 3 (ABC-3), respectively. As previously described, main-
taining a low gastric pH may help nutrient digestion and inhibit the growth of pathogens.
Several researchers reported that a reduction in the pH and/or ABC of the diet, or the addition
of organic acids to the diet, improved animal performance (Partanen and Mroz, 1999; Biagi
et al., 2003). A simple method to measure feed ABC (mEq) is as follows: a 2.55.0 g sample
of feed is suspended in 50 ml of distilled deionized water and left, under continuous agitation,
at 37C for 60 minutes. This is then titrated with 0.1 N HCl or 0.1 N NaOH (depending on
whether pH must be raised or lowered) until pH 3 (ABC-3) or pH 4 (ABC-4) is reached.
Buffering capacity at this point is calculated as:

ABC = {[(50 ML) 0.1] / W} 1000

where W is the weight of the sample and ML represents the volume of 0.1 N HCl or 0.1 N
NaOH needed to reach the desired final pH.
Along with acid-binding capacity, a similar parameter that can be considered is the diet-
buffering capacity calculated as follows: a feed sample (2.55 g) is mixed with 50 ml of HCl
0.1 N and incubated for 1 h in a shaking waterbath at 37C. After that, the pH of the solution
is brought back to 3 by using NaOH 0.1 N. The buffering capacity is then calculated as
follows:

Buffering capacity (mEq/kg) = (50 ml NaOH) 0.1 1000/P

where P = sample weight (g).

As previously described, a low pH in the stomach of the weaning pig is ensured by the pro-
duction of lactic acid and other organic acids (acetic, propionic and butyric acids are the most
important) by microbial fermentation (table 1).
Defining a reliable range of values of the buffering capacity of the diet is still a matter of
conjecture because of the paucity of data relative to single ingredients and their possible inter-
actions. The mineral content and the protein fraction of the diet are the primary factors that
influence ABC (Bolduan et al., 1988). Mroz et al. (2000) suggested that ABC should have a
range of 530600 mEq/kg. Low buffering-capacity diets are reported to improve feed utiliza-
tion and digestibility of nutrients (Blank et al., 1999; Ange et al., 2000; Mroz et al., 2000).
A low ABC diet may help to lower pH in the stomach lumen and allow a proper activation
of pepsin (Taylor, 1959, 1962), leading to a higher gastric digestion of proteins to peptides
and amino acids, which in turn stimulate pancreatic juice secretion (Meyer and Kelly, 1976).
8 A. Piva et al.

Table 1
Amounts of organic acids (mmol/day) produced by microbes in the digestive tract of piglets at
6 weeks of age (source: Jensen, 1998, reproduced with permission of the Institute of Animal
Physiology and Nutrition, Polish Academy of Sciences)

Organic Small Large

acid Stomach intestine intestine Total

Lactic 234 50 266 130 00 500 162

Formic 64 38 20 11 8 55 23
Acetic 42 18 36 15 176 10 254 23
Propionic 41 11 87 5 92 7
Iso-butyric 00 00 60 60
Butyric 23 22 54 3 59 7
Iso-valeric 00 00 61 71
Valeric 11 00 92 10 2
Total 288 68 343 100 350 23 982 124

Even if these results are in agreement with Decuypere et al. (1997), the real relationship
between the ABC of the diet and nutrient digestibility is still under discussion. Moreover, it is
relatively difficult to standardize experimental protocols due to different feedstuff origins, as
well as differences in animal genetics and rearing conditions that characterize animal produc-
tion in the various countries. Nevertheless, the need for safe and natural alternatives to the use
of antibiotics as growth promoters stimulates research in this field.

2.2.1. Lowering gastric pH and buffering capacity

Dietary acidification is gaining more and more interest as it reduces the buffering capacity of
the ingesta, and it may support a more efficient digestion in the stomach resulting in a higher
protein digestibility. Blank et al. (1999) studied the effect of fumaric acid supplemented (0, 1,
2 and 3%) to high and low buffering-capacity diets, calculated according to Bolduan et al.
(1988) (56.7 and 23.5 ml of 0.1 N HCl, respectively), on ileal and fecal digestibility of amino
acids in fistulated piglets. From their findings, fumaric acid exerts a beneficial activity when
added to a diet with low buffering capacity, causing increased ileal digestibility of crude pro-
teins (CP), gross energy (GE) and the majority of amino acids. On the other hand when added
to a high buffering-capacity diet, fumaric acid did not significantly improve any parameter,
although numerical increases in ileal digestibilities of CP, GE and amino acids were recorded.
Biagi et al. (2003), in two in vivo studies in piglets compared six diets: (1) control diet with
plasma protein and carbadox at 55 ppm (PP); (2) plant protein high buffering-capacity diet
(HB); (3) plant protein low buffering-capacity diet (LB); (4) diet 3 plus 1% citric acid
(LB+C); (5) diet 3 plus 1% fumaric acid (LB+F); (6) diet 3 plus 0.2% Tetracid 500 (LB+T;
slow-release organic acids, Vetagro, s.r.l.). Piglets fed diet 1 gained faster (P < 0.05) than those
fed any other diet because of their supplementation with the antibiotic carbadox and plasma
protein. Nevertheless, live weight, average daily gain and feed efficiency did not differ after
4 weeks. At the end of one trial, piglets fed the LB and the LB+T diets weighed, respectively,
9% and 11% more than those fed the HB diet (14.76 and 15.02 vs 13.53 kg; P = 0.10), and
the performance of LB+T fed animals was not statistically different from those of animals on
diet PP where carbadox was present. Interestingly, reducing the buffering capacity of the diet
 Intestinal fermentation: dietary and microbial interactions 9

positively influenced the composition of the intestinal microflora. Thus, there were numerical
reductions of clostridia in the jejunum, and clostridia and coliforms in the cecum even if the
addition of free organic acids to low buffering diets did not influence animal growth or intes-
tinal microflora composition.
The increasing number of data suggesting a modulatory activity of various organic acids on
naturally occurring microflora in the feed before and after ingestion further foster research in
this field. Lrke and Jensen (2003) showed that stomach content from pigs fed a diet supple-
mented with lactic acid (2.2%) had a stable pH below 5 immediately after feeding, while the
stomach content in pigs fed the standard diet displayed more fluctuations and a pH above 5
for up to 2.5 hours post-feeding. Similarly, Jensen et al. (2003) reported that addition of 1.8%
formic acid to the diet of slaughtered pigs stabilized the pH, in the proximal GI tract, below
4 for the whole day, while a non-supplemented diet resulted in pH values of 4.7 shortly after
feeding and in bactericidal (Knarreborg et al., 2002) pH levels (pH below 4) only 4 hours post-
feeding.
The physical form of the feed can also play a role in the extent and efficiency of digestion.
From studies on gastroesophageal ulceration in pigs (Wondra et al., 1995; Regina et al., 1999)
we know that feeding pelleted or finely ground feeds result in a higher incidence of the pathol-
ogy compared to coarsely ground diets. Physical aspects of feedstuffs can affect ammonia and
organic acid production by the gastric microflora. Regina et al. (1999) showed that pigs fed a
finely ground pelleted diet exhibit a higher concentration of ammonia, pepsin and protein in
the stomach, whereas organic acids amounts, namely acetate and L-lactate, were higher in the
stomach of piglets fed a coarsely ground meal. Mikkelsen and Jensen (2003) demonstrated
that a coarse, non-pelleted meal stimulates production of lactic acid as well as that of acetic,
propionic and butyric acids, resulting in a lower gastric pH, and reduced presence of anaero-
bic bacteria. The increased ammonia concentration recorded by Regina et al. (1999) could be
attributed to a microbial pattern dominated by proteolytic bacteria that could metabolize the
highly fermentable form of fine and pelleted feeds.
An interesting approach in lowering gastric pH comes from experiences in feeding fer-
mented liquid feeds (FLF). When fed to piglets, FLF help piglets to overcome the stressful
passage from milk to solid feed, prevent a drastic decrease of feed intake and help mainte-
nance of low gastric pH. Piglets fed FLF have higher concentrations of lactic acid in the
stomach and proximal small intestine as described by Jensen and Mikkelsen (1998) and
Scholten et al. (2002). However, higher concentrations of lactic acid in the stomach do not
coincide with a higher production (mmol/kg/h) of lactic acid in vitro (Jensen and Mikkelsen,
1998; Canibe and Jensen, 2003). This could suggest that most lactic acid in the stomach is
produced from lactic acid bacteria (LAB) fermentations in the feed and not to microbial pro-
duction in situ. Hence the higher concentration of LAB in the stomach should be attributed to
a higher intake with the diet. Even if the studies of Jensen and Mikkelsen (1998) and Canibe
and Jensen (2003) led to similar conclusions, the authors underline the need for new studies
on a larger number of animals in order to improve the statistical power of the results. From
the data discussed, the double presence of a low gastric pH and high number of LAB from
dietary origin seems to be of primary importance. The presence of an already developed lactic
microflora may directly exert its effect in the stomach even if LAB may not colonize that
region. The acidity of FLF coupled with the in situ fermentation of LAB and the production
of lactic acid and other weak organic acids lower the gastric pH. Thus, many enteric bacteria
(Salmonella and E. coli) are killed in the stomach and do not enter the parts of the gastroin-
testinal tract in which they would normally proliferate.
10 A. Piva et al.

2.2.2. Antibacterial mode of action of organic acids

The antibacterial effect of organic acids might be explained by the protons (H+ ions) and
anions (RCOO ions) into which the acid is divided after passing the bacterial cell wall and
which have a disruptive effect on bacterial protein synthesis. There is some evidence from the
literature that fumaric and propionic acid, as well as formic acid, decrease intestinal microbial
growth (Bolduan et al., 1988; Sutton et al., 1991; Gedek et al., 1992).
During their passage through the gastrointestinal tract, prokaryotes like Escherichia coli,
Salmonella typhimurium or Shigella flexneri encounter different and stressful milieu. The
most challenging situation they have to overcome is represented by low gastric pH granted by
the combined actions of weak organic acids from dietary or gastric fermentations and gastric
secretions of HCl. The presence of organic acids seems to be fundamental in preventing
bacterial growth. Dissociation of organic acids follows the HendersonHasselbach equation
(fig. 1), where A and HA are the dissociated and undissociated species, respectively:

pHe = pKa + log[A]/[HA]

The pH value at which molecular acid and dissociated anions are in equal proportions, is
defined as pKa. As shown in fig. 2, organic acids may diffuse across membranes when in the
HA form and then dissociate inside the cytoplasm (Bearson et al., 1997), because of the high
internal pH (pHi), and the anions accumulate (Russell and Diez-Gonzalez, 1998). The conse-
quent drop in pHi, interferes with cellular enzymatic activity, moreover bacterial cells are
forced to reduce their metabolism as energy is primarily required to actively pump protons
outside the cytoplasm. Bearson et al. (1997) described how cells try to raise pHi after milieu
acidification by activation of several amino acid decarboxylases that consume protons (fig. 3).
One example is lysine decarboxylase (CadA) coupled with the lysine-cadaverine antiporter
(CadB) of S. typhimurium. The CadA decarboxylates intracellular lysine to cadaverine and
consumes a proton in the process. Cadaverine is then exchanged for fresh lysine from the sur-
rounding environment via the CadB antiporter (Park et al., 1996). Similar inducible systems,
with arginine and glutamate decarboxylases, have been described for E. coli (Lin et al., 1995).

Fig. 1. Acids rate of dissociation depends on their pKa and on the pH of the environment. As they follow
HendersonHasselbach equation, at neutral pH, there is very little HA, but HA increases logarithmically as
the pH declines. (Source: Piva, 2000.)
 Intestinal fermentation: dietary and microbial interactions 11

RCOOH
pH

pH RCOOH COO

H+

ATP

H+

Fig. 2. Antibacterial mode of action of organic acids: the more lipophylic nondissociated form can permeate
through the bacterial membrane. The higher internal pH (pHi) allows acid to dissociate inside the cytoplasm
(Bearson et al., 1997), and the anions accumulate (Russell and Diez-Gonzalez, 1998). The consequent drop in
pHi interferes with enzymatic activity and cell is forced to reduce its metabolism as energy is primarily
required to actively pump protons outside the cytoplasm and subtracts energy to release protons.

In these mechanisms pH between internal and external seems to be directly involved in

organic acid toxicity, as suggested by Russell and Diez-Gonzalez (1998) with the equation:

pH = log([A] + [HA])in /([A] + [HA])out

hence, the lower the pH the higher the bacterial ability to tolerate organic acid action.
Kajikawa and Russell (1992) observed that passive potassium efflux is a mechanism for
increasing membrane potential and, based on this observation, theorized a potassium-dependent

GABA Agmatine Cadaverine

Glutamate Arginine Lysine

H+ H+ H+
Glut GABA Arg Agmatine Lys Cad

H+ H+ Inducible decarboxylases

H+ H+ RpoS ASPs
MviA

H+ H+ PhoP ASPs Protect/Repair

Macromolecules
H+ H+ Fur ASPs

H+ H+ ? ASPs

Fig. 3. Bacterial mechanisms activated to survive acid shock. Image shows both ATR systems, characterized
by acid shock protein production, and AR systems, based on decarboxylases. (Source: Bearson et al., 1997,
reprinted with permission from Elsevier.)
12 A. Piva et al.

system of pH and membrane potential interconversion. If a bacterium has a very high

concentration of intracellular potassium, membrane potential remains high and pH is low,
and vice versa. This scheme is supported by a contrast between lactic acid bacteria and E. coli.
The lactic acid bacteria, Streptococcus bovis and Lactobacillus lactis, always have very high
internal potassium concentrations and never generate large pH values (Cook and Russell,
1994). E. coli, a bacterium with lower intracellular potassium levels, is able to decrease pH
as the environment becomes more acidic (Kaback, 1990), while potassium addition causes an
almost immediate increase in the intracellular pH of E. coli cells suspended in a medium at
acidic pH (Kroll and Booth, 1983).
A system that could fight acid stress is the acid tolerance response (ATR) (fig. 3). After a
previous exposure to mild acid conditions, the ATR is a complex stress response involving
formation of acid shock proteins (ASP), that permit bacteria like E. coli, S. typhimurium or
S. flexneri to resist in acid environments as low as pH 3, as well as to survive in the presence
of the weak organic acids that usually predominate along the intestine (Bearson et al., 1997).
Audia et al. (2001) reviewed how S. typhimurium ATR induced at pH 4.55.8, allowed the
cells to survive at pH 3 for hours. Guilfoyle and Hirshfield (1996) demonstrated that E. coli
adapted with 11.3 or 13.5 mmol/L of butyrate or propionate at pH 6.5, survive a 30-min chal-
lenge at pH 3.5, whereas Goodson and Rowbury (1989) reported survival at pH 33.5 after
culture in nutrient broth at pH 5. Along with the previously mentioned bacteria, other harmful
pathogens have also been reported to possess ATR systems, and these include: C. perfringens
(Villarreal et al., 2000), L. monocytogenes (ODriscoll et al., 1996), C. jejuni (Murphy et al.,
2003) and H. pylori (Toledo et al., 2002). As such, cells undergoing acid shock in the stomach
will be prepared to endure the environmental stresses in the intestine (Bearson et al., 1997).

3. SMALL INTESTINE
3.1. Morphological changes at weaning

Feeding fermented liquid feeds is also known to increase villi length and to ameliorate the
villus:crypt ratio (Scholten et al., 2002). It is well known that weaning represents the most
critical period in the lifespan of a pig, due to changes in nutritional and environmental condi-
tions and the appearance of new stressors. Pluske et al. (1997) reviewed the different factors
affecting structure and function of the gastrointestinal tract. Burrin and Stoll (2003) described
these changes and divided weaning into an acute phase and an adaptive phase. The most
important factor affecting the acute phase is the reduction of feed intake, and the consequent
decrease in energy supply, due to the learning process a piglet must undergo during the change
to a new feed form (from liquid to solid).
As described by Burrin and Stoll (2003), during the acute phase the intestinal wall experi-
ences a double change: villus atrophy due to an increased cell loss, and crypt hyperplasia
usually indicating an increased crypt-cell production. Hampson (1986) reported that 21 days
after weaning villus height in piglets was reduced to around 75% of that in the preweaning
period, i.e. from 940 m to 694 m. Morphological and functional changes of the intestine
often lead to a reduced intestinal absorption of nutrients that can be metabolized by non-
favorable intestinal bacteria, which in turn can lead to the production of noxious catabolites
or to a possible overgrowth of pathogens. Intestinal changes in response to nutritional,
environmental, sociological and microbiological stimuli have been well documented. As pre-
viously described, the gastrointestinal microflora is a developing organ. At weaning piglets
may easily develop diarrhea (usually within 3 days) usually associated with hemolytic
 Intestinal fermentation: dietary and microbial interactions 13

bacteria such as E. coli. Nabuurs et al. (1993) postulated that the relationship between intes-
tinal structure and scours may stem from the function of villous enterocytes and crypt cells,
since shorter villi and deeper crypts have fewer absorptive and more secretory cells and this
may cause decreased absorption and increased secretion (Pluske et al., 1997). Such a scenario
may induce osmotic diarrhea due to over secretion, and proliferation of hemolytic
E. coli, which may dispose of a higher amount of unabsorbed nutrients.

3.1.1. Nutritional approach

Scholten et al. (2002) tried to overcome villi shortening and crypt deepening by feeding wean-
ing piglets with fermented wheat in liquid diets. Morphological characteristics over 4 and
8 days after weaning revealed longer villi in the first part of the small intestine of FLF piglets,
as the villus/crypt ratio was higher. Moreover, the fermentation products, namely short chain
fatty acids (SCFA), were more favorable for piglets fed FLF. Short chain fatty acids, produced
by microbial fermentation of dietary nutrients, stimulate epithelial cell proliferation both in the
small and large intestines, resulting in a larger absorptive surface (Sakata, 1988). Scheppach et al.
(1992) postulated that normal colonic epithelia derive 6070% of their energy supply from
SCFA, and primarily from butyric acid. The latter induces cell differentiation and regulates
the growth and proliferation of normal colonic mucosa (Treem et al., 1994) while suppress-
ing the growth of cancer cells (Clausen et al., 1991). Piva et al. (2002a) showed in vivo, how
such gut nourishing can affect piglet performances, reliably affecting small intestinal
mucosa. The study was conducted using 40 weaned piglets divided into two homogeneous
groups, fed a conventional nonmedicated diet without (CTR) or with sodium butyrate (SB) at
0.8 g/kg. Both diets were also supplemented with formic and lactic acid at 0.5 and 1.5 g/kg
of feed, respectively. The beneficial effects of butyric acid were appreciable in the first period
of the study (014 days) with higher average daily gain (ADG) (+20%; P < 0.05) and higher
daily feed intake (+16%; P < 0.05). A higher feed intake was also recorded during the second
phase (1535 days) although it was not associated with a higher ADG. This loss of feed effi-
ciency is most likely connected to an effective response of the intestinal architecture to SB
only during the first phase (014 days). Conversely, in the following period SB might have
stimulated feed intake without stimulating an equally effective utilization of nutrients. The
improved growth performance could be associated with the beneficial effect of butyric acid on
the proliferation of the intestinal epithelium. This is of greater biological value during the
weaning period when the weight of the small and large intestine increases three times faster
than that of the (whole) body mass growth (Sakata and Setoyama, 1997). It must be consid-
ered that the supplied amount of butyric acid (5 mol/g DM feed) could have been of
biological significance only for the small intestine where baseline values for butyric acid are
about 4 mol/g DM. Conversely, cecal concentrations of butyric acid are of about 240 mol/g
DM (Piva et al., 2002b). As such, even in the unlikely event of the entire amount of SB reach-
ing the hindgut, the addition of SB at the tested dose would have had no influence on
colonocyte metabolism. This, in turn, substantiates why the efficacy of SB is limited to the
post-weaning period, when the villus structure is more negatively affected by the transition to
solid feed and when it may benefit from the growth modulation effect of SB (Hodin et al.,
1997). Other studies have shown positive effects of butyric acid on ileal villi and cecal crypt
structure (Galfi and Bokori, 1990; Piva et al., 2002b).
As reviewed by Burrin and Stoll (2003) and from their own experiences, a large proportion
of dietary nutrients are preferentially metabolized by the gut in the so-called first-pass metab-
olism. Some of these, namely glutamine, glutamate and SCFA, are of particular importance
14 A. Piva et al.

as energetic substrates for enterocytes. After ingestion, only 10% of dietary glutamate, gluta-
mine and aspartate appear in the portal flow, indicating a large utilization by the portal drained
viscera (Stoll et al., 1999). Measuring this usage it appears that a high proportion of each of
these three non-essential amino acids is oxidized to CO2 (5070%), whilst the remainder is
converted to lactate, citrulline, ornithine, arginine and alanine.

3.2. Intestinal amines

Along with glucose, other metabolic fuels for the small intestine are represented by the natu-
ral polyamines: putrescine, spermidine and spermine. These natural amines are fundamental
for the proliferation and cellular evolution of living cells. Heby and Persson (1990) reported
that there was an interruption of cell division in cell cultures lacking the ability to produce or
absorb polyamines. From a biochemical point of view these biogenic amines are polycations,
positively charged at physiological pH, that may form bridges between negative charges on
the cell membrane to stabilize cell functions (Tabor and Tabor, 1984; Pegg, 1986; Osman et al.,
1998). Moreover, they may act as second messengers interacting with DNA and RNA struc-
tures as well as with protein metabolism (Heby, 1981; Pignata et al., 1999; Wallace, 2000).
Polyamines in mammalian cells are mainly formed by decarboxylation of ornithine to
putrescine, by the enzyme ornithine decarboxylase (ODC). Putrescine is then converted to
spermidine by the enzyme spermidine synthase and consequently spermine is formed from
spermidine due to the action of spermine synthase. Therefore, synthesis of these last two
polyamines needs the presence of S-adenosylmethionine decarboxylase. The interconversion
pathway is catalyzed by the enzyme spermidine or spermine acetyltransferase. As described
by Dufour et al. (1988) and Bardocz et al. (2001) polyamines play key roles in intestinal mat-
uration and development in the young animal. The body-pool of these polycations is used
according to the needs of the different regions of the body and the amount of newly absorbed
or produced compounds (White and Bradocz, 1999). The presence of these amines in entero-
cytes, is ensured by three sources: (1) lumenal polyamines; (2) circulating blood pool; and
(3) newly synthesized inside the cell.
Luminal pool polyamines originate from the diet, defoliated cells, pancreatic secretions and
bacterial metabolism. The contribution of bacterial flora is still under continuous debate and
is not well understood or well described. Bardocz et al. (2001) summarized the contribution
of de novo biosynthesis (14 moles/d/100 g rat), diet (16 moles/d/100 g rat) and intestinal
microflora (34 moles/ d/100 g rat) to the body polyamine pool in the rat. Even though
Bardocz et al. (1993) analyzed over 40 food ingredients and reported that high quantities
(hundreds of micromoles) should enter the human gut lumen every day, the real pattern of
polyamines and even biogenic amines (cadaverine, histamine, tyramine) characterizing host
intestinal lumen is still nebulous. Moreover, interactions with different types of diets and feed
additives and the role of bacterial polyamine production is unknown. In a recent study, Piva
et al. (2002b) reported on mono-, di- and polyamine (table 2) contents in the jejunum and
cecum of piglets fed a control diet with or without the addition of tributyrin and/or lactitol. As
a nondigestible oligosaccharide, lactitol showed the ability to modulate intestinal microflora
and reduce proteolysis (Piva et al., 1996a), whereas tributyrin is thought to be a dietary source
of butyric acid for the gut. The study showed that there were no alterations of the physiolog-
ical level of polyamines (Bardocz et al., 2001) within the small intestine, even though the
intestinal wall was positively affected as indicated by morphometric measurements. Moreover,
bacterial production of SCFA was shifted towards a significantly higher production of lactic
acid, showing a positive enhancement of lactic acid bacterial activity. Interestingly, only animals
Table 2
Mono-, di- and polyamines (mol/g DM) in the jejunum and cecum of piglets fed a control (CTR) diet with or without tributyrin (TRB) and/or lactitol (LCT)
(source: Piva et al., 2002b, reproduced with permission of the American Society of Animal Science)

Organic acid

Tyramine Cadaverine Histamine Putrescine Spermidine Spermine

Jejunuma
CTR 2.04 1.01 1.21 0.60 2.81 0.39b 0.68 0.28 0.36 0.08 1.06 0.12
TRB 1.18 0.62 1.76 1.18 2.45 0.31bc 0.68 0.33 0.38 0.10 1.58 0.96
LCT 1.97 0.51 1.63 0.36 2.90 0.35b 0.78 0.08 0.43 0.12 1.33 0.28
TRB+LCT 1.83 1.06 1.67 0.41 0.95 0.32c 0.98 0.31 0.54 0.12 0.88 0.21
Cecuma
CTR 1.34 0.20 4.32 1.46 2.97 0.39b 4.25 0.71 1.37 0.23 1.02 0.14
TRB 0.87 0.24 4.39 1.97 1.66 0.17bc 4.46 1.96 1.40 0.33 0.93 0.16
LCT 1.54 0.31 5.31 1.04 2.31 0.19bc 4.55 1.02 1.52 0.14 0.97 0.21
TRB+LCT 0.72 0.25 2.98 1.24 1.51 0.41c 3.41 1.12 2.14 0.12 0.91 0.26
a
Values are means SE, n = 4. b,cValues in the same column and in the same intestinal site with different superscripts are different (P < 0.05).
Intestinal fermentation: dietary and microbial interactions
15
16 A. Piva et al.

fed both tributyrin and lactitol showed a lower concentration of histamine in the small and
large intestine. Histamine, released by mast cells, exhibits various biological effects, related
to allergic enteropathy, inflammatory bowel disease (Raithel et al., 1995), and stress-related
gut dysfunction (Santos et al., 1998). To counteract these noxious effects and to avoid passage
of this diamine into the systemic blood circulation, the intestine usually degrades it, as well
as cadaverine, by activation of diamine oxidase. This enzyme is also necessary for the oxida-
tion of putrescine to -aminobutyric acid (GABA) allowing its action as a growth factor (Seild
et al., 1985). These considerations are important in young developing animals, which have an
immature gut and very low diamine oxidase activity, so that a high concentration of histamine
or cadaverine may reduce the effective oxidation of putrescine (Bardocz et al., 2001), and
consequently delay gut maturation.
The roles played by other biogenic amines, namely tyramine and cadaverine, are still
unresolved. Lyons et al. (1983) indicated that cadaverine may enhance histamine toxicity by
inhibiting histamine metabolism, which leads to increased uptake of nonmetabolized hista-
mine. They did not, however, support the hypothesis that potentiation occurred via an overall
increase in the absorption of histamine and its metabolites due to some disruption in the bar-
rier function of the intestine. On the other hand, cadaverine in humans has been shown to exert
beneficial effects in containing Shigella flexnery. It induces compartmentalization of Shigella
species to the phagolysosome, which constitutes a protective response of the host that directly
contributes to the diminished ability of polymorphonuclear leukocyte-rich inflammations to
transmigrate across model intestinal epithelia (Fernandez et al., 2001). Similarly, Kohler et al.
(2002) investigated whether piperidine, a cadaverine metabolite, could be used against infection
with enteric pathogens. They demonstrated that piperidine treatment prevented the invasion of
S. typhimurium into model intestinal epithelium by nearly 95%. In vivo studies revealed that
piperidine treatment lowered the death rate in mice infected with S. typhimurium and reduced
bacterial translocation and colonization of various organs and tissues.
Moreover, Bermudez and Firman (1998) studied biogenic amines in broilers as they were
implicated in causing poor performance and intestinal lesions in chickens. They fed animals
with phenylethylamine (4.8 mg/kg feed), putrescine (49 mg/kg feed), cadaverine (107 mg/kg
feed), histamine (131 mg/kg feed), or a combination of all these amines. Recorded parame-
ters at 2, 4 and 6 weeks included performance, gross lesions and histology. The authors did
not observe any consistent effects on performance by any of the treatments, nor were gross
lesions observed on a consistent basis and no histopathological remarks were reported.
These and other reports represent an open dilemma on the roles of these amines, their
dietary origin and metabolism, their influences on intestinal microflora and whether enhanc-
ing or diminishing their concentration may have positive or negative influences on host
performance and welfare.

4. LARGE BOWEL
Intestinal fermentation occurs mainly in the hindgut (Decuypere and Van der Heyde, 1972),
where decarboxylation of several amino acids by bacteria can produce monoamines (tyramine
and tryptamine from tyrosine and tryptophan, respectively) and polyamines (putrescine and
spermidine from arginine and ornithine) (Dierick et al., 1986).
The intestinal microflora is also deeply involved in the production of ammonia. Ammonia
is produced both by endogenous and bacterial enzymes within the alimentary tract. Bacterial
enzymes appear to produce 75% of the alimentary tract ammonia, with urea hydrolysis being
the major contributor in mammals residing in conventional, nongerm-free environments
 Intestinal fermentation: dietary and microbial interactions 17

(Visek, 1984). Energy is the limiting factor for fermentation in the hindgut (Orskov et al.,
1970). As energy sources (starch and fermentable carbohydrates) are depleted, the fermenta-
tion becomes more and more proteolytic. This results in ammonia and amine production
(Russell et al., 1983). Ammonia can destroy cells, alter nucleic acid synthesis, increase intes-
tinal mucosal cell mass, increase virus infections, favor growth of cancerous cells over
noncancerous cells in tissue culture (Visek, 1978), and it can reduce the villus height
(Nousiainen, 1991). Furthermore, absorbed ammonia must be excreted as urea at an energy
cost of approximately 7% of the total energy expenditure in monogastric as well as in rumi-
nant animals (Eisemann and Nienaber, 1990). High plasma concentrations of ammonia may
inhibit insulin release to a number of stimuli, impairing glucose metabolism and animal
performances; whilst high protein diets create an environment in the reproductive tract
characterized by high pH, that can reduce the vitality and motility of sperm (Visek, 1984).
At the same time, bacterial fermentation of dietery fiber may produce large quantities of
SCFA, that are readily absorbed by the colonic mucosa. As previously reported, short chain
fatty acids play a key role as energy sources; butyric acid being the most quickly oxidized to
CO2 among all the SCFA in the intestine (Fleming and Gill, 1997). Butyric acid has also been
shown to induce cell differentiation and to regulate the growth and proliferation of normal
colonic and ileal mucosa (Treem et al., 1994), whereas it can actively reduce the growth rate
of colorectal cancer cells (Berry and Paraskeva, 1988).
As the origin of SCFA is mainly from nondigestible polysaccharides, modulation of these
nutrients is likely to influence the distribution and concentration of SCFA in the
ileal/cecal/colonic lumen. For decades, the pig industry has been utilizing the crude fiber
method of Weende. Today, the more accepted method for referencing dietary fiber is the
approach developed by Van Soest in 1967 in which the fibrous fraction is described by three
components: neutral-detergent fiber (NDF), acid-detergent fiber (ADF) and acid-detergent
lignin (ADL). These components play key roles in the digestion process by modulating the
viscosity of the diet and its transit time through the gastrointestinal tract, the water-holding
capacity, digestibility and cecal fermentation. For example, Berggren et al. (1993) suggested
that dietary supplementation with guar gum may lead to a higher production of propionic acid
whilst pectins are related to a dominant release of acetic acid (Brighenti et al., 1989). The
main factor affecting fiber metabolism is fermentability and transit which together can deter-
mine the time available for bacterial fiber degradation. Henningsson et al. (2002) described
how different sources of dietary fiber fed alone or in combination, may vary the SCFA pattern
in rats. They investigated the fermentability and pattern of SCFA derived from rat large bowel
metabolism of highly fermentable indigestible carbohydrates, i.e. guar gum (GG), pectin
(Pec) and high amylose corn starch (HAS) or resistant fibers like wheat bran (WB), fed
singularly or in combination. Pectin released the highest proportion of acetic acid (76 2%
of total SCFA) and GG of propionic (31 4%; P > 0.0005) but they lowered butyric acid
production. Interestingly, when fed in combination the butyric acid pool was doubled (9.0 1.1%
vs 16.6 3.3%; P > 0.05). Incorporation of WB delayed the site of fermentation of HAS to
the distal part of the hindgut. Bach Knudsen et al. (1994) also showed probable species
differences in the capacity of cecal microflora to utilize different fiber sources.
Taking these suggestions into consideration, the possibility of modulating cecal fermenta-
tion through novel feed additives or diverse feed components metabolized through elective
microbial pathways to release the desired SCFA takes on ever more relevance. Moreover, even
if it is still highly debated, the most accredited mode of action of antibiotic growth promoters,
seems to be an action on the hindgut microflora, that leads to the establishment of beneficial
bacterial populations (lactic acid bacteria), i.e. that may alter intestinal metabolism to a more
18 A. Piva et al.

beneficial pattern for the host. A desirable dietary formulation should result in low gas pro-
duction, which may determine gut bloating and the so-called abdominal pain with consequently
reduced feed-intake, reduced ammonia concentration and high levels of SCFA. On the other
hand, Gaskins (2003) raised considerable doubt that antibiotics could work also against the
so-called beneficial bacteria leaving more nutrients available for the host.
In fact, even if the positive influences of microbially produced SCFA are well known, it is
still under discussion whether bacterial metabolism and nutrient transformations may be of
value for the host. Over the years, two different kinds of bacterial populations have been
described in the literature: the beneficial commensal bacteria (lactic acid bacteria) and the
potentially harmful bacteria (coliforms, clostridia, salmonellae). Usually, commensals are
described as providing nutrients such as SCFA, vitamins and amino acids, while in addition
they confer some protection from pathogens by competitive exclusion. Conversely, as
reviewed by Gaskins (2003) the host spends relevant energies trying to keep microbes away
from the epithelial surface (pathogens and nonpathogens alike), and to quickly start-up
inflammatory and immune responses against those organisms that pass the mucosal defenses.
In a previous work, Anderson et al. (2000) concluded that host and microbiota are in compe-
tition for nutrients in the small intestine, whilst in the hindgut they are in symbiosis because
of the final products of fermentation of indigestible feed components. Strategies directed
towards ameliorating gut microbial mass, enhancing only beneficial bacteria, pose a double
paradox since this increases mucosal metabolism while limiting dietary nutrient availability.
The question posed by Gaskins (2003), whether energetic contributions of SCFA to whole
animal metabolism are more important than their use for maintenance of a voluminous
cecum-colon densely populated by fermenting bacteria, is still open and unresolved.
Moreover, it seems that microbial manipulation may improve a specific bacterial population
compared to others, but gastrointestinal stability is better served by a stable diversity.
Traditional culturing techniques are often limited in studying changes in the microecology of
the GI tract, because of the difficulties related to growth of anaerobes, and appropriate selec-
tive media. The development of molecular techniques based on 16S rRNA genes, is now
applicable to the complex intestinal environment (Vaughan et al., 2000). Favier et al. (2003)
using a PCR-DGGE based method, described changes in intestinal bacteria of 60 piglets
weaned at 21 days of life and sampled at 0, 2, 5, 8 and 15 days post-weaning. Their results
confirmed the presence of deeply unstable microbial communities during weaning, mostly in
the period between 5 and 8 days when all but one of the species detected as different gel bands
seemed to disappear. Interactions between diet and bacterial changes are still not well under-
stood due to the difficulties in approaching these subjects.
Different topics have been investigated both in vivo and in vitro, in order to evaluate non-
conventional feed additives, spanning across organic acids, prebiotics, probiotics, symbiotics
and botanicals. Since what was previously described relates to variations in microbial popu-
lations, trials on new strategies usually take into account and analyze indirect parameters of
the activity of new additives on the gut ecosystem, such as SCFA production, total gas pro-
duced during fermentation, ammonia and amine production, etc.

4.1. In vitro system

An in vitro fermentation system was developed (Piva et al., 1996b) in order to properly inves-
tigate the relationships between diet, microbes and potential natural additives. With such an
approach it is possible to study fermentation parameters over 24 48 h, either in the small or
large intestine and, by using at least 30 fermentation vessels, a statistically correct evaluation
 Intestinal fermentation: dietary and microbial interactions 19

of dietary ingredients or additives at various inclusion rates can be made. Such a strategy is
preliminary to an in vivo study and it narrows down the most interesting solution to be inves-
tigated in vivo. The method is based on two main steps to simulate ileal digestion, as described
by Vervaeke et al. (1989): (1) predigestion of the basic feed diet (2 g; particle size < 1 mm),
with an incubation in 40 ml of pepsin solution (2 g/L, HCl 0.075 mol/L) at 39C for 4 h;
(2) the pH is adjusted to 7.5 with NaOH (1 mmol/L), 40 ml of pancreatin solution (10 g/L in
phosphate buffer pH 7.5) is added, and the mixture is incubated in a shaking water bath at
39C for 4 h. After enzymatic digestion, the preparation is centrifuged, washed three times
with distilled water and dried at 55C overnight.
Fermentation is carried out in a batch culture system using the cecal contents from several
slaughtered animals, pooled, filtered and diluted with buffer (McDougall, 1948) (ratio 1:2),
before dispensing into fermentors. Samples are then taken for SCFA and ammonia analysis,
while gas production is measured as described by Menke et al. (1979) using syringes with the
same liquor collection procedure, the same volume of liquor and the same predigested feed
concentration as the fermentation vessels. Gas production is referred to as an index of micro-
bial metabolism, and so data are interpolated on the Gompertz bacterial growth model,
assuming that substrate levels limit growth in a logarithmic relationship (Schofield et al.,
1994). The Gompertz equation for gas production is as follows:

V = VF exp {exp [1 + (me/VF) ( t)]}

where symbols have the meaning assigned by Zwietering et al. (1990): V = volume of gas
produced at time t, t = fermentation time, VF = maximum volume of gas produced, m =
maximum rate of gas production, which occurs at the point of inflection of the gas curve and
= the lag time, as the time-axis intercept of a tangent line at the point of inflection.
The duration of the exponential phase is calculated as the difference between the time point
where the third derivative of the growth model becomes zero for the second time, and the lag
time. The duration of the exponential phase can be calculated from the parameters of the mod-
ified Gompertz equation, as suggested by Zwietering et al. (1992) with the following:

exponential phase (h) = VF /(me){1 ln [(3 5 )/2]}

4.2. Organic acids

The addition of organic acids to the diet has been already described relative to their potential
ability in lowering the buffering capacity of the ration. Lactic acid bacteria are usually not
influenced by their presence, whilst coliforms, salmonellae and clostridia are the more
targeted bacterial strains, so that inclusion of these compounds in the diet may modulate the
fermentation process in the hindgut. Even if extensively studied, real organic acid activity
inside the gastrointestinal tract remains controversial. Using the above-described in vitro
system, Biagi (2000) screened 11 different organic acids: formic, acetic, propionic, lactic,
butyric, sorbic, fumaric, malic, citric, -ketoglutaric and benzoic acid, at three different con-
centrations (60, 120 and 240 mmoles/L fermentation liquor). The organic acids influenced
cecal fermentation and their effects varied depending on the acid and its concentration. When
the acids were used at 60 mmoles/L, only sorbic acid was able to reduce the total volume of
gas produced (VF), compared to control (34%), while citric acid and -ketoglutaric acid
increased VF compared to control, by 92% and 32%, respectively. With acids at 120 mmoles/L,
VF was reduced by sorbic acid and benzoic acid, by 34% and 49%, respectively, whereas
20 A. Piva et al.

lactic acid, citric acid and -ketoglutaric acid increased VF by 74%, 52% and 40%, respec-
tively. When used at 240 mmoles/L, lactic acid still increased VF by 35% compared to control.
Compared to control, ammonia concentrations at 8 h were reduced by lactic acid at
60 mmoles/L (29%) and by sorbic acid at 240 mmoles/L (27%). The same ammonia-
lowering effect was observed at 24 h for lactic acid, fumaric acid, -ketoglutaric acid and
benzoic acid at 120 and 240 mmoles/L. On the contrary, acetic acid and malic acid at
60 mmoles/L, acetic acid, butyric acid and malic acid at 120 mmoles/L, and formic acid, acetic
acid and butyric acid at 240 mmoles/L produced higher ammonia concentrations than control.
These findings suggest that organic acids can positively influence cecal fermentation
in a dose-dependent manner, and that sorbic and benzoic acids are the most effective in reduc-
ing total gas and ammonia production. Benzoic acid was also reported to be effective in
reducing coliforms in the stomach (Knarreborg et al., 2002). Other acids, such as citric acid,
-ketoglutaric acid and lactic acid, boost cecal fermentation, probably acting as an energy
source for some cecal microflora strains, increasing total gas production or gas production rate
and decreasing ammonia concentrations.
When fed to weaning piglets, organic acids have been tested extensively to achieve specific
targets (e.g. pH lowering, bacterial inhibition). In vivo effects on microbial populations are
dose dependent and usually visible at high concentrations (Jensen et al., 2003). Thus, lactic
acid has a positive effect on yeast and lactic acid bacteria at doses between 0.7% to 2.8%
while significantly reducing coliform counts (Maribo et al., 2000). Similar high concentra-
tions were proposed as necessary by Tsiloyiannis et al. (2001) testing different acids in
postweaning diarrhea piglets affected by ETEC strains. Because at high doses organic acids may
be detrimental for operators and machinery, a coating could be applied. Moreover, the adoption
of a strategy of microencapsulation can result in the slow release of coated compounds along the
intestine (Piva et al., 1997a), affecting microbial metabolism throughout the intestine.
Partanen (2001) showed in vivo how low doses of single SCFA (< 25 g/kg) may positively
affect growth performances in weaned piglets. Her meta-analysis of the published data
reported significant (P < 0.05) improvements of average daily gain and feed to gain ration in
animals fed acidified diets.
Another reliable strategy implies the use of organic acid blends, which take advantage of
the synergistic effect of certain acids allowing administration of lower doses in the diet. Piva
et al. (2002c) evaluated in vitro, at pH 6.7, the effects of adding a commercial blend of organic
acids (Tetracid500, Vetagro, Italy) providing phosphoric acid, citric acid, fumaric acid and
malic acid at 1.53, 0.78, 2.59 and 1.12 mmol/L of fermentation liquor, respectively) to three
diets with: 0 (low fiber, L-NDF, neutral detergent fiber; Van Soest et al., 1991), 100 (medium
fiber, M-NDF), and 200 g/kg (high fiber, H-NDF) of dried sugar beet pulp.
Replacing 10% or 20% of the L-NDF diet with sugar beet pulp increased the NDF dietary
level and resulted in an increased volume of gas produced (VF) and rate of gas production
(m). The above information supports an increased availability of fermentable energy by
increasing the NDF level of the diet, as also suggested by the shorter time required to reach
the inflection point of the gas production curve. It seems that the stimulatory effect of sugar
beet pulp could be accounted for by the soluble fraction (e.g. pectins) escaping NDF deter-
mination. When added to L-NDF, the acid blend resulted in an increased maximum rate of
gas production. This finding could be explained by the fact that citric acid (Lutgens and
Gottschalk, 1980; Marty-Teysset et al., 1996), malic acid (Renault et al., 1988; Loubiere et al.,
1992) and fumaric acid (Tran et al., 1997; Tielens and Van Hellemond, 1998) may positively
modulate the energy metabolism of some bacterial strains usually residing in the hindgut. Lopez
et al. (1999) observed that sodium fumarate at 5 and 10 mmol/L was able to stimulate ruminal
 Intestinal fermentation: dietary and microbial interactions 21

proliferation of cellulolytic bacteria and digestion of fiber. In this study, the use of a blend of
organic acids at low concentrations did not stimulate fiber digestion as indicated by the low
concentration of acetic acid (Stewart and Bryant, 1988). Instead, the lower concentrations of
ammonia, iso-butyric acid and iso-valeric acid in the vessels containing the organic acid blend
provide an indication of effective control of the proteolytic process by the organic acids even
after 24 h of fermentation. The above isoacids are formed from the deamination of valine and
leucine (Van Soest, 1982) and are indicative of the extent of protein catabolism. Iso-butyric
and iso-valeric acids, although in limited amounts, are extremely important as they are growth
factors for many cellulolytic organisms and other species that can use them for long chain
fatty acid synthesis and for amino acid synthesis through reverse reactions (Van Soest, 1982).
Since fiber fermentation by cellulolytic bacteria leads generally to acetic acid production
(Stewart and Bryant, 1988), the poor availability of isoacids could explain the significant reduc-
tion of acetic and n-butyric acids that we observed in the vessels containing organic acid blends.

4.3. Prebiotics

Another category of molecules that can play a role as microbial modulators are the prebiotics,
defined as nondigestible food ingredients that beneficially affect the host by selectively
stimulating the growth and/or activity of one or a limited number of bacteria in the colon
(Gibson and Roberfroid, 1995). There are several categories of fermentable substrates that can
act as prebiotics, including nonstarch polysaccharides (NSP; Shi and Noblet, 1993), dietary
resistant starch (Jacobasch et al., 1999), nondigestible oligosaccharides (NDO; Piva et al.,
1996a; Houdijk et al., 1997), and milk whey (Piva et al., 1998).
Several types of NDO are currently available: fructo-oligosaccharides (FOS), gluco-
oligosaccharides (GOS), mannano-oligosaccharides (MOS), galacto-oligosaccharides (GAS),
xylo-oligosaccharides (XOS). They may derive from plant origins (FOS and GAS), from enzy-
matic polysaccharide hydrolysis (FOS and XOS) or from de novo synthesis (FOS, GOS, GAS).
The use of prebiotics is aimed at enhancing beneficial bacteria (Bifidobacterium,
Lactobacillus) inside the gut, by nourishing them with preferential substrates. The degree of
selectivity of such NDO for certain types of bacteria is still under discussion. As bifidobacte-
ria do not produce hydrogen or carbon dioxide, fermentation by bifidobacteria does not result
in gastrointestinal distension and abdominal pain. Unfortunately, some prebiotics have been
shown to result in gas overproduction, which may limit their usage (e.g. lactulose and -
galactosides; Levrat et al., 1991). Hartemink and Rombuts (1997) described the capability of
intestinal bacteria to ferment NDO (table 3).
Even with this approach, an in vitro fermentation system may help to identify the best can-
didate for in vivo studies. An extremely interesting NDO tested in vitro and in vivo is lactitol.
Lactitol is a disaccharide which consists of galactose and sorbitol with a -galactoside bond.
This sugar alcohol is only poorly absorbed in the small intestine (Dharmaraj et al., 1987) and
reaches the hindgut where it is fermented (Nousiainen and Setl, 1992). Jensen (1993) sug-
gested an intriguing antiproteolytic effect of this sugar-alcohol, with reductions in
deamination of amino acids and ammonia production, when lactitol is present in the lower gut.
Piva et al. (1996a) conducted a study to determine if the response of swine cecal microflora
to lactitol (-D-galactopyranosyl-(14)-D-sorbitol), varies when fermenting low-fiber (LF)
or high-fiber (HF) predigested diets. The inoculum was collected from four sows fitted with
cecal cannulas, pooled, buffered and dispensed in 27 vessels under anaerobic conditions.
Lactitol (L) significantly lowered end pH and the acetic to propionic acid ratio in the first
8 hours of experiment (P < 0.05 and reduced ammonia by 100% and 84% in LF+L and
 22

Table 3
Fermentation of NDO by selected intestinal bacteria (source: Hartemink and Rombouts, 1997, reproduced with permission of Wageningen University)

Bacterial group/species FOSa INU TOS GLL IMO RAF LAT LAC PHGG

Bacteroides distasonis
B. fragilis
B. ovatus
B. thetaiotaomicron
B. vulgatus
Bifidobacterium spp.
Clostridium butyricum
Cl. clostridioforme
Cl. perfringens
Cl. ramosum
Escherichia coli
Eubacterium lentum
+ +
+ +
+ +
+ +
+ +
+ +
+,
+, ,+
+ +
,+
+ + + +, + +
+ + + +, + +
+ + +, + +
+ + +, +
+ + + +, +
+ + + + + +,
+ + + +
+, +
,+ + +, + +
+ +, + +
+ +,
A. Piva et al.

Eu. limosum
Fusobacterium
necrophorum
Lactobacillus +, + + + +, + +
acidophilus
group
Lb. casei +, + + + +
Megasphaera elsdenii
Mitsuokella multiacidus +, + + + +
Ruminococcus. productus +, +
Veillonella parvula
a
FOS: fructo-oligosaccharides; INU: inulin; TOS: trans-galactosyl-oligosaccharides; GLL: 4-galactosyl-lactose; IMO: isomalto-oligosaccharides; RAF: raffinose; LAT: lactulose; LAC: lactitol;
PHGG: partially hydrolyzed guar gum.
 Intestinal fermentation: dietary and microbial interactions 23

by 56 and 38% in HF+L diets (P < 0.05) at 4 and 8 h, respectively. In addition, LF+L and
HF+L diets gave higher SCFA energy yields by 70% and 40% than LF and HF, respectively
(P < 0.05). Two bacterial growth models (logistic and Gompertz) were tested to fit the gas pro-
duction data and of these, the Gompertz equation provided the best fit. Lactitol reduced
culture lag time by approximately 50% and increased gas production rate and maximum gas
production by 60%, but only when the microflora was fermenting the LF predigested diet
(P < 0.05). These data indicate a key role of lactitol in driving the hindgut metabolism to a
better usage of nonstarch polysaccharides and eventually an increased availability of SCFA
for the host. The efficacy of lactitol in containing the presence of ammonia in the LF diet and
hence avoiding proteolysis would appear to confirm this hypothesis. In a subsequent study
Piva et al. (1997b) confirmed these results and also showed that lactitol has the capacity to
reduce indole and skatole, two L-tryptophan catabolites with detrimental effects on animal
health and meat quality (Lundstrom et al., 1994; Henry, 1995).

4.4. Combo strategies

Although different approaches (organic acids, NDO) have shown beneficial influences, as
alternatives to antibiotics, in modulating the fermentation process within the gastrointestinal
tract when supplemented alone, evidence is growing for the efficacy of an intriguing new
approach. It seems that a combination of more than one novel approach may lead to an even
more favorable equilibrium of intestinal metabolism and thus animal welfare and perfor-
mance. Literature concerning this strategy is still weak, even though some trials have been
carried out. This approach takes into account all the different aspects of the GI tract: micro-
biology, nutrient metabolism and tissue requirements.

4.4.1. Pro + pre-biotic = synbiotic

The combination of a probiotic and a prebiotic can be a synergistic strategy that beneficially
affects the host by improving the survival and the implantation of a direct-fed microbial in the
gastrointestinal tract, and by electively stimulating the growth and/or by activating the metab-
olism of a limited number of health-promoting bacteria (Roberfroid, 1998). The beneficial
response can be more evident when animals are challenged by pathogens or chemicals. Ziprin
and DeLoach (1993) found a further reduction of intestinal colonization by Salmonella in
chicks by administering lactose to animals that had already received probiotic cultures.
Similarly, the combination of bifidobacteria and oligofructose reduced colon cancer risk in
carcinogen-exposed rats (Gallaher and Khil, 1999).
Piva et al. (2005) analyzed a symbiotic effect first in vitro and then in vivo on weanling
pigs. After screening to select the best combination of lactic acid bacteria and the already
promising prebiotic lactitol (Piva et al., 1996a, 1997b) two synbiotics were selected: lactitol
+ Lactobacillus brevis P6 4/9 and lactitol + Lactobacillus salivarius 1B 4/11. The improved
beneficial effects of these associations were evident by reductions of ammonia production at
8 h (10.82 and 9.81 vs 11.99 mmol/L, respectively; P < 0.05) and at 24 h (9.92 and 9.24 vs
12.85 mmol/L, respectively; P < 0.05) compared to lactitol alone, suggesting that a properly
selected synbiotic can be more effective than the prebiotic component alone in controlling
proteolysis. Moreover, reduced proteolysis may also be implied from the in vivo results.
Plasma urea levels were higher in the treated groups. Rychen and Nunes (1995) described an
increase of amino-nitrogen in the portal vein after feeding a probiotic to young pigs and
supposed that this could be the effect of stimulating endogenous proteolytic activity, or the
24 A. Piva et al.

consequence of an improved absorption of free amino acids in the intestinal lumen. Moreover,
the synbiotic enhanced SCFA production, and hence higher energy yield, in the hindgut as
observed in vitro. The better intestinal fermentation parameters resulted in an improved feed
efficiency in vivo (+15%, P < 0.05).

4.4.2. Prebiotic + gut nutrient

As the intestine represents a complex environment, trying to promote the intestinal ecosystem
may be best achieved through manipulation of nutrient availability and microbial activity.
Following this concept, application of probiotic cultures, alone or in combination with
prebiotic oligosaccharides, has been found to ameliorate microbial population patterns in the
gastrointestinal tract and, in so doing, favorably affect the host (Howard et al., 1993; Tannock,
1999). There have also been a few reports about the development of flavorings and herbal
extracts for stimulating appetite, as well as for displaying antagonism toward undesirable
microbes and improving the antioxidant status of the host and, in so doing, beneficially affect-
ing the health status in swine or poultry (Luchansky, 2000; Piva, 2000).
After in vitro studies on lactitol (Piva et al., 1996a, 1997b), Piva et al. (2002b) investigated
tributyrin and lactitol (a prebiotic) as dietary and fermentable sources of butyrate, respectively,
(US patent 6,217,915). This approach couples the needs of modulating intestinal bacteria to
produce positive SCFA and at the same time supports the tissues by directly nourishing them
with specific nutrients. The 28-day-old piglets in the study were fed a common commercial
diet (CTR) with or without tributyrin (TRB), lactitol (LCT) alone or in combination
(TRB+LCT). Compared to animals fed the control, tributyrin or lactitol diets, animals fed the
TRB+LCT diet displayed the most desirable outcomes for all of the parameters measured.
These animals experienced no weight loss and no mortality during the 42-day feeding period.
These animals also showed an improved ADG and feed efficiency, and achieved a 34% higher
total live weight at the end of the study than animals fed the control diet (237.4 vs 176.8 kg
for the TRB+LCT and control groups, respectively).
Tributyrin+lactitol decreased histamine production in both the jejunum and cecum. The
release of histamine by mast cells exhibits various biological effects related to allergic
enteropathy, inflammatory bowel disease (Raithel et al., 1995) and stress-related gut dysfunc-
tion (Santos et al., 1998). Histamine lowers the blood pressure by dilating blood vessels and
causes inflammatory reactions by promoting leukocyte chemotaxis (Mitsuoka, 1993).
Histamine is also associated with increased colonic secretion (Wang et al., 1990) and ileum
contraction (Bartho et al., 1987), as well as with celiac disease by inducing atrophy of villi,
hyperplasia of crypts and increase of mucosal volume (Wingren et al., 1986). As such, feed-
ing the TRB+LCT diet may be beneficial by limiting the exposure of the gut to
proinflammatory conditions. Moreover, small intestinal nutrition was positively affected, as
judged by villus height and crypt depth. The mucosal structure with longer villi and shorter
cecal crypts observed in animals fed the lactitol or the TRB+LCT diets supports the hypoth-
esis that nutrient absorption in the small intestine is best with the least energy-demanding
configuration for the hindgut.

5. CONCLUSION
Intestinal fermentation varies dramatically due to the complex interactions between three fac-
tors: digestive tract development and nutrition, diet composition and digestibility and bacterial
composition and metabolism. Such interactions evolve during the life span of the host as well
as across the various sections of the gastrointestinal tract.
 Intestinal fermentation: dietary and microbial interactions 25

The change in the consumers demand for a safe food production chain and the recent reg-
ulatory issues about the ban of antibiotic growth promoters have ensured not only a search for
natural strategies to modulate gut development and health, but also a much deeper under-
standing of the above-described interactions.

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